It is well known that deoxyribonucleic acid (DNA) is a helically-shaped double stranded molecule having complementary base pairs connecting the two phosphate-sugar backbone strands. The ability to cleave DNA or RNA, both single strand and double strand, is important. It is particularly important that it be possible to direct this cleavage such that it occurs at specifically-defined sites in the DNA or RNA molecule. Such DNA and RNA cleavage agents could be useful in a laboratory setting for biological or pharmaceutical research or as part of a diagnostic tool. It is also possible that such agents could be used in vivo as part of a gene therapy regimen, for example in the treatment of cancer.
At the present time, there are several types of reagents that can be used to cleave DNA either thermally or photochemically. These include (1) dienyne natural products and their synthetic derivatives; (2) other reagents that generate biradicals, e.g., azoalkenes; (3) organoplatinum reagents; (4) anthraquinones; and (5) rhodium and ruthenium complexes. The dienyne products and their derivatives are very complex molecules that will cleave DNA either thermally or photochemically. This DNA cleaving activity is thought to be the source of their activity against certain types of cancer. These compounds, however, are not readily available. The need to isolate these materials from their natural sources makes it difficult to obtain dienynes in large quantities at reasonable prices. Even if these compounds were synthesized chemically, the synthesis would involve multiple step reactions just to prepare the active molecules, not to mention the additional synthetic effort that would be necessary to attach them to DNA sequence specific groups. Thus, the dienyne materials do not offer a cost-effective way of cleaving DNA molecules.
Azoalkenes that form trimethylenemethane biradicals (see Bregant, T. M.; Groppe, J.; Little, R. D.; J. Am. Chem. Soc. 1994, 116: 3635-3636) are thermally unstable and decompose over several days at room temperature.
Anthraquinones are para-quinones that are highly reactive and sensitive to visible light. They will destroy their own sequence-recognition chains unless stored in the cold and rigorously protected from visible light.
Organoplatinum reagents have also been shown to be effective for the photochemical cleavage of DNA. Here again, these reagents are relatively expensive to produce in bulk quantities and are not particularly amenable to the synthetic modification which is necessary if DNA sequence recognition properties are to be incorporated into them. Furthermore, the toxicological properties of these materials can become a problem when used in vivo, for example in the treatment of cancer.
Rhodium and ruthenium complexes have also been shown to be effective as photochemical agents for the cleavage of DNA. However, like the organoplantinum complexes, they are expensive to manufacture and could pose significant toxicological risks if they are to be used in vivo.
There is, therefore, a need for DNA cleavage reagents which are easy to synthesize in bulk quantities, which are relatively stable on storage and will survive to reach their target site in the cell, which do not involve the cost, availability or toxicological issues present with the precious metal compounds, and which may easily be modified to bind to specific DNA sequences and, therefore, cleave DNA at specifically defined sites. It is this problem which the present invention seeks to address.
Dervan and Becker, J. Am. Chem. Soc. (1978) 100: 1968-1970, describes the synthesis and use of bis(methidium) spermine as a polyintercalating molecule (i.e., a molecule which simultaneously binds to adjacent sites of DNA). The approach taken in this work was to form a dimer of ethidium bromide, a known intercalating molecule, using spermine as the linking group. The molecules described in this paper are not taught to be useful to cleave DNA.
Morii, et al, J. Am. Chem. Soc. (1993) 115: 1150-1151, studied the sequence- specific DNA binding of certain peptide dimers and found that certain enantiomers bind better than others. The molecule used to link the sequence-recognizing monomers was 9,10-dihydrophenanthrene-9, 10-diol, a planar moiety. The molecules described in this paper are not taught to be useful to cleave DNA.
Armitage, et al, J. Am. Chem. Soc. (1994) 116: 9847-9859, studied the use of certain para-quinone compounds, i.e., amide and ammonium-substituted anthraquinones, as photocatalytic DNA cleaving agents.
Sitlani, et al, J. Am. Chem. Soc. (1992) 114: 2303-2312, studied phenanthrenequinone diimine complexes of rhodium (III) and concluded that they promote DNA cleavage in the presence of ultraviolet light.
U.S. Pat. No. 4,699,978, Barton, issued Oct. 13, 1987, describes bis-substituted ruthenium or cobalt metal complexes of phenanthrolines which bind stereospecifically to DNA and can be used to cleave DNA in the presence of ultraviolet light.
U.S. Pat. No. 5,258,506, Urdea, et al, issued Nov. 2, 1993, deals with a particular family of chemicals which are suitable for being incorporated into oligonucleotide chains so as to introduce sites which may be cleaved by exposure to ultraviolet light. The materials disclosed in this patent are not, themselves, used to cut naturally occurring DNA strands at desired sites. Rather, they would be incorporated into the DNA molecules of interest via hybridization, and the resulting complex would be cleaved at the site at which the materials are situated.
U.S. Pat. No. 5,162,218, Schultz, issued Nov. 10, 1992, describes a means for attaching a polypeptide to a binding site to provide an active functionality at that site. The functionality allows reporter molecules having therapeutic or catalytic activity to bind at the site.
Pfundt, et al., Tetrahedron 22(7):2237-2247 (1966) [abstract at Chem. Abs. 65: 10474a (1966)], describes the determination, by NMR spectroscopy, of the conformation of various 1,4-dioxene derivatives. There is no suggestion that these compounds could be used in cleaving DNA.
The Arnitage, et al and Sitlani, et al papers and the Barton patent all disclose compounds useful in the photocatalytic cleavage of DNA. These molecules all incorporate planar moieties in order to achieve intercalation. However, none of these disclosures utilize the "masked quinone" approach of the present invention which allows the molecule to embed itself in and cleave DNA, but not react with other cell components. The Pfundt, et al., article which describes compounds similar to those used in the present invention, does not suggest their use in a biological context.